Screening of Process Parameters for Aspirin Loaded PLGA Microsphere

Introduction

Aspirin (Acetyl Salicylic Acid, ASA), the wonder drug can regulate a variety of disease conditions and has the potential to stimulate multiple dental stem cells, thereby promoting the regeneration of dentin and the periodontal ligament.^1,2 According to the study conducted by Rahman et al., aspirin has the capacity to stimulate the periodontal ligament stem cells and helps in the stimulation of growth factors like fibroblast growth factor (FGF), Vascular Endothelial Growth Factor A (VEGFA) and Bone Morphogenetic Protein (BMP) and aids in osteogenic potential.² The study also revealed that aspirin at concentration 1000 µM is capable of enhancing the osteogenic potential, and is non-cytotoxic to the stem cells. At this dose, the drug is capable of enhancing the activation of biological functions, cellular signalling and cellular growth, proliferation and differentiation. These findings strongly imply that aspirin has excellent potential in the management of several dental problems for which there is no defined treatment strategy.

Furthermore, different biomaterials (such as chitosan, PLGA, alginate, etc.) offer physical, chemical, and biological environments to control cell responses for growth and differentiation.³ They are efficient carriers of nutrients, oxygen, and waste and thus are a platform for cell growth and development of new tissues. This three-dimensional structure substitutes for the extracellular matrix, which releases the growth factor involved in neo-tissue formation.⁴ The incorporation of bioactive molecules improves the interaction with cells and enhances migration, differentiation, and proliferation of dental cells, consequently enhancing tissue regeneration.^3,4 According to previous studies, polylactic glycolic acid (PLGA) can promote proliferation, osteogenic differentiation, initiation of mechanical support, and formation of three-dimensional niches for new tissue growth, as well as inhibit the early down-growth of gingival epithelium and connective tissues.^5,6 Consequently, the denuded root surface can be repopulated by regenerative cells. Various researchers have formulated various forms of PLGA like films, microspheres, nanoparticle and scaffolds, to incorporate the biological markers for the regeneration of various dental tissue structures.^7-11 Therefore, it is necessary to investigate the therapeutic effectiveness of aspirin by designing and developing a novel PLGA formulation.

However, the effective development of appropriate formulations necessitates taking into account several variables that have an impact on the formulation's performance, such as the physicochemical properties of the drugs and excipients, their composition, and various manufacturing processes.¹¹ These parameters can be screened by selecting a suitable experimental design. Therefore, the Plackett-Burman experimental design facilitates the determination and assessment of independent variables that influence the formulation response by mathematically calculating the significance of a large number of factors in one experiment.¹² In the current study, the Plackett-Burman design was used to examine the effects of process parameters on the synthesis of aspirin-loaded PLGA microspheres, and significant process parameters that primarily influence encapsulation efficiency were identified.

Materials and Methods

Aspirin and PLGA (75:25) were a gift sample from Alta Labs, and Evonik, India. Ethyl acetate (S.D. fine chemicals, India), Poly (vinyl alcohol) (Mw 1,30,000, 18-88% hydrolyzed, Sigma-Aldrich) were procured. Analytical grade solvents were used.

Formulation of Aspirin loaded PLGA microsphere

The double-emulsion solvent extraction method was adopted to formulate the aspirin-PLGA microsphere.^12,13 PLGA and aspirin were dissolved in the organic phase (acetate-ethanol solvent system), and an aqueous solution was injected. The mixture was emulsified at varying speeds and different time interval using a homogenizer (Kinematica Polytron™ PT2100) to produce a waterin-oil emulsion (w/o). The resulting w/o emulsion was injected (at different flow rates) into a polyvinyl alcohol solution (PVA, different concentrations) and emulsified at room temperature to form a W/O/W emulsion. The formed microspheres were hardened using the solvent extraction method by dissolving the excess aqueous phase and stirring in a magnetic stirrer overnight. Finally, the samples were centrifuged (15,000 rpm for 10 min) and washed continuously to separate the microspheres. Isolated microspheres were screened and optimized using suitable statistical experimental method.

Experimental design and analysis

In this study, the Plackett-Burman screening design was used to investigate the effects of four process parameters, including homogenization time (min), stirring speed (rpm), emulsification time (min), and flow rate (mL/min), as well as four formulation variables such as PVA concentration (%), volume of the internal phase of the primary emulsion (mL), solvent system (ethyl acetate: ethanol), and PLGA concentration (%) on the encapsulation efficiency of the microspheres. Design-Expert® Version 12 was used to devise a 12- run, eight-factor Plackett-Burman screening design (Stat-Ease Inc., Minneapolis, USA). Factors were assigned a "high" and a "low" level, and are presented in Table 1. To estimate the variance (experimental error) of an effect, insignificant dummy variables were assigned in addition to the variables of real interest. These variables were not assigned any values.

Determination of Encapsulation Efficiency

The difference between the amount of aspirin added during formulation and the amount of unentrapped drug in the external phase following formulation was used to calculate the encapsulated efficiency (EE). The aspirin that was not encapsulated was checked in the supernatant by UV spectroscopy at 277 nm after centrifugation of the formed suspension for 10 minutes at 15,000 rpm.⁹

Results and Discussion

Screening Design

Screening designs aim to identify formulation variables that can be modified or eliminated for further studies. Plackett-Burman screening was used to consider the main effects among the eight independent variables. The variable levels were considered based on preliminary experiments and data not shown.

The response of the encapsulation efficiency obtained is recorded in Table 2, and varies between 50.11% and 88.98%.

Data were transferred into Design Expert (12.0) software, and a linear model for the encapsulation efficiency of aspirin in terms of coded factors was developed as shown in equation.

Encapsulation efficiency = 39.58167-0.730667A-0.006 993B+4.29333C-1.72500D+0.031893E-1.2F+ 17.42G-0.295111H

From the above equation and the model's P-value, F-value, mean square, and R2 (from the ANOVA shown in Table 3), it can be deduced that the PVA concentration, volume of the internal phase, and PLGA concentration have a significant impact on encapsulation efficiency. The statistical significance of the Plackett Burman design can also be examined using standardized Pareto charts (Figure 2), which represent the estimated effects of the parameters on the response. The t-value of Student’s t-test, which is represented by the length of the bars, is proportional to the absolute value of the estimated effects divided by the standard error.

Furthermore, improved encapsulation efficiency due to PLGA and PVA concentrations may be due to increased viscosity, which can prevent the drug from diffusing from the organic phase.¹⁴

In addition to this positive effect of PLGA concentration, larger particles may be formed when PLGA concentration is increased. Hence, it provides a sufficient surface for aspirin entrapped.^14,15

The second prominent effect is the PVA concentration, which accounts for an increase in viscosity due to concentration, preventing the outward diffusion of the drug from the internal aqueous phase and better stabilization of the double emulsion at higher PVA concentrations.¹⁰

Conclusion

The Plackett-Burman screening design was devised to study the favorable effect parameters on the encapsulation efficiency of aspirin-loaded PLGA microspheres. Twelve experimental runs were conducted to establish the relationship between the factors and the response. PLGA concentration, volume of the internal phase, and PVA concentration, significantly affected encapsulation efficiency. The screened factors were used for further optimization of aspirin-PLGA microspheres.

Conflict of interest

None